Two mechanisms assure that T lymphocytes are not self reactive: central and peripheral tolerance. Central tolerance (or negative selection) is imprinted during T cell development in the thymus, when self antigen reactive thymocytes are signalled for apoptosis. At the same time, positive selection operates to spare only cells that are MHC restricted. The interactions of the T cell receptor with its MHC/peptide ligand dictates both positive and negative selection. Yet the cellular context of these two selective steps is also critical. This application explores what molecular interactions, in addition to the T cell receptor, are needed during negative selection in the thymus. Preliminary experiments showed that thymocytes can become activated or die, in vitro, depending on both the nature of the TCR ligand and the cellular context. To define the minimal requirements for negative selection, we will use microspheres as antigen presenting units. Various candidate molecules will be adhered on microspheres together with the MHC/peptide complexes that have varying affinities for the TCR, and presented to thymocytes in vitro. The proposed experiments will define the molecules that costimulate death in thymocytes. The possibility that thymocyte activation without death in vitro represents the first step in positive selection will be approached by transferring the in vitro activated cells into the thymii of host animals and following their fate. Thymocytes undergo death in vivo at two district stages of maturation. A fetal thymic organ culture model of early and late deletion will be established to ask if affinity is the central factor that determines the stage of death. Various antagonist reagents will be used to block early versus late deletion. This will allow a definition of the differential requirements for costimulatory molecules at each stage. Finally, microspheres that dicate different outcomes in thymocytes (life or death) will be used to study the biochemical signal involved. Three different areas of potential signal diversity will be explored, based on the knowledge of the intracellular signals delivered in peripheral T cells. These experiments may allow us to understand how life and death are dictated in thymocytes. Our studies of central tolerance will complement the studies of peripheral tolerance by other on this program project, with the broader aim of understanding immunological self tolerance.